Celiac antigen

ABSTRACT

In this invention a novel antigen and a novel antibody-antigen complex are provided for screening patients suspected of having celiac disease. The antigen is prepared by starting with human placenta tissue which is perfused with a hepes and collagenase buffer to obtain a single cell suspension. This suspension is enriched to obtain an enriched protein portion and then separated out to obtain an embryonic celiac antigen (ECA). This ECA is used with serum from patients to effect binding of ECA with IgA in the serum while applying a human IgA antibody to the serum. The results are then read on a spectrophotometer to confirm or negate the presence of celiac disease.

This invention relates to a novel antigen and, more specifically, to anovel antigen useful as a screening assay for celiac patients. Thisapplication is a continuation-in-part of application Ser. No.09/018,419, filed Feb. 4, 1998 for Novel Celiac Antigen, now abandoned,which in turn is a divisional application of application Ser. No.08/626,243, filed Mar. 29, 1996 for Celiac Antigen, now U.S. Pat. No.5,716,794.

BACKGROUND OF THE INVENTION

Several known antigens are known and are presently being used foridentifying celiac disease in both adults and children. Some of theseantigens and the screening procedures are outlined in the articles“Precipitins to antigens of wheat and cow's milk in celiac disease” byHeiner D C, Lahey M E, Wilson J F, et al., published in 1962 in J.Pediatr. 61,814; “A reliable screening test for childhood celiacdisease: fluorescent immunosorbent test for gliadin antibodies. Aprospective multicenter study,” published in 1983 in J. Pediatr.102:655-60; “Gliadin antibodies in celiac disease” published in 1983 inJ. Pediatr. 102:711-2. There are also several other papers andpublications concerned primarily with tests for detecting celiacdisease. While some tests have been found to be somewhat effective andat least sometimes accurate, there is a need for a more reliable antigenand uniform interpretation of screening assays for celiac patients.

Childhood Celiac Disease (CD) is a condition characterized bymalabsorption and growth disturbances in association with a specifichistological lesion of the small intestine. Patients with malabsorptivesymptoms have been found to date to the second century AD. In 1950'sDicke, a pediatrician found the relationship between grain consumptionand severe malabsorption condition. He noticed that during WWII childrenin Holland with malabsorptive symptoms improved when wheat and rye flourwere unobtainable and that the condition reappeared after wheat flourwas again made available. He and his associates were also credited withidentifying gluten, the water-insoluble protein fraction in wheat, asthe toxic dietary substance responsible for the syndrome, noting thatsymptoms subside in response to gluten elimination.

Recently, the European Society for Pediatric Gastroenterology andNitrition (ESPGAN) revised the criteria for the diagnosis of glutensensitive enteropathy (GSE), celiac disease. The previous criteria setforth in 1969 required at least three small intestinal biopsies. Thesalient histological features of CD were required at the initialsuspicion of the enteropathy. The histological findings were expected toresolve following a period of gluten elimination and be reinduced duringa gluten challenge phase of establishing the diagnosis. Since then, thesuccess of newer diagnostic a markers—antigliadin, antireticulin andantiendomysial antibodies—in serving as indicators of active disease hasprompted a revision of the criteria. Current requirements include acharacteristic histologic appearance of the mucosa at the time ofpresentation with resolution of symptoms following gluten elimination.The presence of the previously mentioned circulating antibodies at timeof diagnosis and disappearance following gluten withdrawal adds weightto the diagnosis especially in those who are asymptomatic.

Few controversies in the discipline of Pediatric Gastroenterology areviewed with less emotion that the selection of the best serologicscreening test for celiac disease. The discovery of circulatingantibodies to gliadin (AGA), reticulin (ARA) and the endomysium (EMA)have brought us closer to the discovery of a simple non-invasivescreening test for establishing the diagnosis; but none are universallyaccepted as being pathognomonic indicators of the condition. Althoughnone can be considered, as yet, pathognomonic, the serologic antibodiescan still be used for screening and aid in diagnosis if one realizestheir limitations and interprets their presence or absence in light ofthe clinical situation faced. We are approaching a better understandingof their roles and interrelationships.

The above mentioned present day serological markers are circulatingantibodies which serve as one piece of evidence for the immunologicalnature of the disease. Circulating antigliadin antibodies (AGA)represent antibodies to the cereal protein, which presumably is absorbedintact across the intestinal mucosa. AGA have been extensivelyinvestigated since initial descriptions appeared in the late 1950's andearly 1960's. Techniques for detection have evolved over the yearsvarying from precipitating antibodies to cereal proteins,microimmunodiffusion, radioimmunoassay, binding serum antibodies towheat grains and detection by fluorescent horse antihuman IgG. The firstELISA method appeared in 1977 and is defined and described in detail inHekkens W T, Van Twist M: Physiological role of antigliadin antibodiesand their appearance in celiac disease; Chorzelski T P Beutner E H,Kuman V, Zalewski T K, eds. Serologic Diagnosis of Celiac Disease.Cleveland:CRC Press, 1990: 2A: 21058 and Hekkens W T, VanLems-Kan P H,Rosekrans PCM: Bepaling van gliadin antistoffen met de ELISA-Techniek.Ned Tijdschr Geneesk 121, 1908, 1977. The differences in techniques fordetection led to problems with standardization and thereforereproducibility between studies. Several authors have attempted toimprove the sensitivity of the method of detection by using a diffusionto gel ELISA as described in Lindberg T, Nilsson L. Borulf S, et al.:Serum IgA and IgG gliadin antibodies and small intestinal mucosal damagein children. J. Pediatr Gastroenterol Nut 4, 917, 1985. It isunderstandable that even similar techniques such as ELISA may yielddifference results because gliadins are a complex mixture of proteinsthat contain at least 40 different components for a single variety ofwheat. Several authors have attempted to improve the sensitivity ofdetection method by using gliadin fractions or peptides as antigens. Inone study, different gliadin peptides were found to have differingreactivity to serum antibodies of untreated celiac patients. However,other authors report that there are no demonstrable differences in ELISAvalues using different gliadin fractions as antigens. Discordantresults, therefore, have been reported despite the use of similarmethodologies such as ELISA.

Further controversy exists regarding the value of the specific class ofAGA antibodies in the diagnosis of celiac disease. Some investigatorsadvocate IgG class AGA whereas the IGA-AGA are favored by others. Instudies using purified gliadin peptides as antigens in RIA or ELISA, IGAantibodies seem to have greater specificity to celiac disease and IgGantibodies seem to be more sensitive. The usefulness of gliadinantibodies for diagnosis, however, is open for criticism because theirsensitivity and specificity varies so much from study to study. Figuresfor specificity range between 65-100% for IgA antibodies and 50-100% forIgG antibodies. Sensitivity reports for IGA antibodies range between52-99.9% and for IgG between 82-100%. Unfortunately, IgG antibodies canbe found in normal controls as well as other disease controls such asChrohn's disease, liver disease and other G.I. disorders. IgAantibodies, on the other hand, although being specific for celiacdisease, are not found in all celiacs. Additionally, IgG are found toincrease with age in normal controls making them unsuitable fordiagnosis in older age groups. The combined use of the two classes ofantibodies, therefore, has been proposed by some authors as improvingthe sensitivity and specificity of the AGA for diagnosis. At least onerecent report did not find advantage in combining each. The merit ofusing the IgG AGA seems to be in its ability to discriminate those 3% ofceliac patients who are also IgA deficient and therefore would not beidentified by screening for IgA AGA.

Gluten challenge is followed by an increase in titres of both IgA andIgG-class antibodies and titres fall with gluten exclusion. IgGantibodies take longer to decline than IgA antibodies. They may takemore than six months to decline whereas IgA antibodies decline by 2-6months. Their appearance is stated to occur before symptoms becomeovert. IgA AGA antibodies are therefore suitable to monitor dietarycompliance and also response to gluten provocation.

Antireticulin antibodies (ARA), on the other hand, have beeninvestigated since initial reports in the 1970s. The ARA were firstdescribed as antibodies reacting with connective tissue fibers aroundhepatic sinusoids, and blood vessels as well as perilobular,periglomerular and occasional glomerular staining of the kidney and alsofor fine staining of the stroma between gastric glands. ARA reacted withconnective tissue of both rat and human organs and appeared to bedirected against reticulin fibers in these tissues. The antibody wasbest detected by indirect immunofluorescence using rat liver and kidneyas the substrate. The ARA do not react with type III colagen,noncallagenous reticulin components or fibronectin and they seem to bespecific for other unidentified connective tissue components. Severalstaining patterns by indirect immunofluorescence were found to occur. AnR 1 pattern was exhibited by celiac and dermatitis herpetiformispatients. This pattern was characterized by staining of peritubular andperiglomerular fibers in the kidney and fluorescence in portal tracts ofrat liver. In contrast to other ARA subtypes, it also reacted with humantissues, although some authors disagree to the human expression. Theyinsist that the reticulin antigen are specifically expressed in rodentbut not in primate tissues. ARA can be of the IgG or IgA class but IgMantibodies do not occur. IgG antibodies usually occur in conjunctionwith IgA ARA. The specificity of IgG-ARA for GSE is controversial. TheIGA-class ARA seem to be disease specific and sensitive indicators ofgluten-sensitive enteropathy. The range of specificity reportedly liesbetween 59-100% and sensitivity between 30-95% indicating somewhatsimilar sensitivity and specificity as the IgA-AGA, however, someauthors may argue that the R-1 ARA are probably more specific for celiacdisease than the gliadin antibodies. However, the R-1 ARA also have beenreported in patients with Crohn's disease and occasionally with otherconditions. The significance of the R-1 ARA is uncertain, however, theassociation between it and untreated celiac disease is well establishedand it will disappear from the circulation following the start of astrict gluten-free diet. Sensitivity and specificity reports are subjectto differences in populations studied. For example, some exclusivelylooked at specific populations such as India, where one does not expectto find patients with inflammatory bowel disease, the authors found thatR-1 ARA were 100% specificity with 85% sensitivity.

A third group of circulating tissue antibodies, the endomysialantibodies (EMA), are gradually gaining acceptance as sensitive andspecific markers of celiac disease. These are primarily IgA antibodiesdirected against the intermyofibril substance of the smooth muscle whichmay correspond either to a reticulin-like structure or a surfacecomponent of smooth muscle fibrils. This antigen may also be expressedaround lamina propria structures around intestinal crypts, muscularismucosa and smooth muscle fibers to which “human jejunal antibodies” aredirected. Unlike R-1 ARA which is reported to react with human andvarious rodent tissues, EMA has been demonstrated to bespecies-specific, reacting only with the endomysium in thegastrointestinal tract of primates. They are detected by indirectimmunofluorescence using monkey esophagus tissue sections. Thesensitivity and specificity of the EMA approaches but is not 100%. Somefalse positive cases have been reported. A case of cows' milk proteinallergy and one with Giardia lamblia have been reported. In addition,one celiac patient was discovered who was IgA and IgG EMA negative onpresentation and during gluten challenge. Therefore, we believe thatfalse negative cases are less common that false positive cases but thatthey do exist and will continue to be reported. Absence of EMA may bemore frequent in celiac patients younger than two years than in olderindividuals. In a recent report, three children with positive and fourwith weak positive results did not have celiac disease. Sensitivity wasfound to be 100% with specificity at 97%. In another study whichcompared EMA, AGA and ARA in an Israeli group of celiac patients,specificity was 98% and sensitivity was 97%. In this study, the EMAappeared to be the most reliable serologic marker for the diagnosis ofceliac disease. The positive predictive value of the EMA and ARA werecomparable (97% and 100%, respectively), however, the EMA had thehighest negative predictive value (98%). Additionally, EMA were found tobe more diet sensitive. By three months of gluten withdrawal morechildren were negative for EMA than AGA or ARA. High EMA titres appearquicker in response to gluten challenge. No serologic test at presenttherefore enjoys a 100% sensitivity and specificity. However, of all themarkers, we believe the EMA is presently the best serologic test basedon our interpretation (bias, if you will) of currently available data.

The occurrence of false positive and false negative results with theabove markers requires some comment. Deficiencies exist with theuniformity of interpretation of the methodologies of detection of theabove antibodies using indirect immunofluorescence. When one encountersreports of false negative cases in using indirect immunofluorescence,one is tempted to question the technique and interpretation of the testsystems. Also, what titre should be considered positive? Should weaklystained sections at low serum dilution be considered positive? One alsoneeds to consider whether the patient is IgA deficient when IgA, AGA,ARA or EMA are being used. Indeed, some investigators advocate that IgAAGA do not offer advantage over IgG AGA and that subjects with isolatedselective IgA deficiency and celiac disease would be better picked up byscreening for both IgG-AGA and IgG-ARA since IgG-AGA is less specificand are commonly found in non-celiac subjects with selective IgAdeficiency.

When one discovers false positive cases for EMA or either AGA or ARA,one wonders whether these may be latent celiacs. Truly, the antibodiesare demonstrated in these cases but why they are present remains anenigma. Maybe one should not consider this as “false” positive. Onlyfollow-up studies will determine whether these individuals developceliac disease. Indeed, this type of situation has been recentlyaddressed. Seven of 25 patients exhibiting ARA or AGA and having normalsmall-bowel mucosal villous architecture at presentation were found tosubsequently develop villus atrophy after a follow-up period of 1-7years. Patients who are “false” positive for EMA may also prove todevelop the histopathologic features of celiac disease in the future.Future studies will elucidate this issue. With regard to false negativecases, however, there seems to be very few published cases of celiacswho do not possess the EMA.

Since it is becoming generally accepted that subclinical celiac diseaseis common in the general population, the use of screening tests inclinical studies is become increasingly important. Many patients who arefree of major symptoms are being identified to possess the typicalhistological features of Celiac Disease. They are discovered duringstudies addressing the incidence of celiac disease using serologicmarkers as screening tests in high risk populations such as familymembers, patients with diabetes, short stature or in the generalpopulation. When one considers the silent celiacs, the true incidence ofthe disease in the USA and Europe may be higher than reported. It wasfound using EMA that the rate in family members was 8% and those withdiabetes was 4% and short stature 1.7%. These are similar figures tothose reported in European studies. However, the incidence in patientswith major symptoms was only 1.29 per 10,000 live births which is muchlower than the mode of 1:1,000 reported in European centers. Studies arebeing awaited with great interest that address the incidence of silentceliacs in both populations. Certainly, the incidence of those withmajor symptoms is much less than Europe.

The efforts to discover the best screening test for celiac disease andefforts to diagnose every last celiac have vastly overshadowed andoutnumbered studies of the pathophysiology of the disease. While gliadinantibodies were report some 30 years ago, work on characterizing thetoxic component(s), both in vivo and in vitro, has not been completed.The molecular mechanism behind the pathogenesis of CD is still unsettledbut the immunological aspects of the disease have attracted greatattention both in the proposal of pathogenetic mechanism and in theefforts to establish a serodiagnostic test for CD.

The different gliadin polypeptides can be separated into four groups,the alpha, beta, gamma and delta fractions by electrophoresis at acidicpH. The molecular weight of a toxic gliadin in the gamma fraction hasbeen recently reported to lie between 35-45 kd. Many hypothesis havebeen proposed to explain the pathomechanism of the observed gliadininduced enteropathy. Studies have been limited to investigations oflymphoctye populations and cytokine production or toxic peptides actingas lectins which induce cell death. These seem promising, serve todirect future studies at immunologic reactions associated with tissueinjury but definitive statements regarding pathophysiology cannot yet bemade. Furthermore, the mechanism of generation of the antigliadin isuncertain. There are even less reports which address the reticulin andendomysial antibodies. One expects that the identification andcharacterization of the antigens to which these antibodies are directedwill help in understanding the pathomechanism of the gluten inducedenteropathy. It is hoped that future work will be directed atpathophysiology; not only identification and diagnosis. It is expectedthat characterization of the above antigens will aid in understandingthe pathophysiology of the disorder and may even lead to a betterunderstanding of the above diagnostic tests.

SUMMARY OF THE INVENTION

It is therefore an object of this invention to provide a novel antigenuseful in screening tests for Celiac Disease devoid of the above noteddisadvantages.

A further object of this invention is to provide a novel antigenprepared from human placenta for use in a method to identify CeliacDisease.

Another object of this invention is to provide a substantially reliableantigen having protein molecules of specifically defined molecularweights which when isolated are useful in a screening process fordetecting Celiac Disease.

Still a further object of this invention is to provide a novel andsubstantially more accurate antigen than those previously used inscreening patients suspected of having Celiac Disease.

These and other objects of this invention are accomplished, generallyspeaking, by a novel antigen which in one preferred embodiment isisolated by a method including the identification, isolation andproduction of an antigen starting with a portion of human placenta. Thisplacenta is perfused using a hepes and collagenase buffer, enriching theresulting composition by condensing it and extracting protein therefrom.The protein is then isolated and prepared for use as an antigen toidentify Celiac Disease. This antigen will bind to the IgA/IgG containedin the sera of patients with Celiac Disease. This novel antigen isdesignated as Embryonic Celiac Antigen (ECA).

Solid phase immunoassay techniques are commonly used to detect thepresence of antibodies that react with specific antigens to form anantibody-antigen complex. In such complexes, the binding between theantibody and antigen typically involves one or more of various weakforces, such as, hydrogen bonds, van der Waals forces, electrostaticinteractions, and hydrophobic interactions. The formation of anantibody-antigen complex may be demonstrated by further reaction of thecomplex with a labeled anti-immunoglobulin antibody. In the commonlyused enzyme-linked immunosorbent assay (ELISA), the detection involves acolor change resulting from the further addition of a secondary antibodylabeled with a suitable enzyme, such as, horseradish peroxidase or analkaline phosphatase. The color change is conveniently detectable byspectrophotometric techniques. It has been found that when an antigen ofthe present invention (ECA) is reacted with the sera of a celiac diseasepatient, a novel antibody-antigen complex is formed that serves as anindicator of the presence of that disease. Thus, the complex as well asthe method of formation provide method of screening for celiac disease.

The ELISA technique is well known and is described in detail in thefollowing publications: Hekkens, W T, Van Twist, M: Physiological roleof antigliadin antibodies and their appearance in Celiac Disease. InChorzelski T P, Beutner E H, Kumar V, Zalewski T K, eds. SerologicDiagnosis of Celiac Disease. Cleveland: CRC Press, 1990: 2A: 21-58; andHekkens W T, VanLems-Kan P H, Rosenkrans PCM: Bepaling van gliadinantistoffen met de ELISA-techniek. Ned Tijdschr Geneesk 121, 1908, 1977.This ELISA technique will be used as the methodology for utilizing ECAantigen and a novel antibody-antigen complex formed therefrom, as ascreening test for Celiac Disease.

SUMMARY OF THE INVENTION

Thus, this invention provides:

(1) a novel antigen (ECA) to be used in screening for Celiac Disease;and

(2) including a novel method for preparing the novel ECA. Disclosed alsoherein is a novel antibody-antigen complex and a novel screening processusing ECA as the novel antigen.

The antibody-antigen complex of this invention is prepared by a processcomprising contacting (1) an embryonic celiac antigen isolated byextraction from a cultured human placenta cell, comprising proteinmolecules with an isoelectric point (pI) range of from 5.1-5.8 and anapparent molecular weight selected from the group consisting of 55 Kd,65 Kd, and 110 Kd, with (2) sera from a celiac disease patient. Thepreferred antigen comprises protein molecules with an apparent molecularweight of 65 Kd.

This novel ECA when used in a screening process will replace the EMA andARA (indirect Immunofluorescence) procedures above discussed. Theaccuracy of detecting Celiac Disease using ECA alone or with otherprocedures is increased about 15% to 20% over prior celiac detectingprocedures which is considered very substantial. The placenta that isused is any available human placenta, but the embryonic fetal portion ofthe placenta is used as the starting material. It is critical to thisinvention that the eventual protein that is isolated and prepared foruse as the antigen has an isoelectric range, i.e., A pI of 5.1-5.8 and amolecular weight of 55 Kd, 65 Kd and 110 Kd or mixtures of these threemolecular weights. Those proteins having a different molecular weightdid not function as a reliable antigen for Celiac Disease. Competitionbinding assay was used to identify the separated proteins. These threesingle polypeptides with molecular weights of 55 Kd-65 Kd and 110 Kd andIsoelectric point 5.1-5.8 demonstrated inhibition of binding of CeliacDisease patient serum IgA/IgG on the monkey esophagus and ratkidney/liver. Seven other monopolypeptides with molecular weight of lessthan 50 Kd inhibit binding to Gliadin.

In a preferred embodiment, the novel antigen of this invention isisolated by the following method:

a) cleaning and washing a human placenta tissue with HEPES buffer;

b) digesting the said placenta tissue with collagenase buffer to obtaina placenta cell population in suspension, said placenta cell populationcontaining cytotrophoblast cells;

c) enriching the cytotrophoblast cell population in said suspension viaa 0-30% discontinuous Percoll gradient;

d) extracting protein from said enriched cytotrophoblast cells withlysing buffer, extraction buffer and via ultracentrifugation to obtain aprotein portion;

e) isolating embryonic celiac antigen (ECA) from said exacted protein byapplying said protein portion to an ion exchange DEAE Sephadex columnfollowed by size exclusion gel chromatography; and

f) identifying by SDS-PAGE and western blotting embryonic celiacantigens comprising protein molecules with an isoelectric range (pI) offrom 5.1-5.8 and an apparent molecular weight selected from the groupconsisting of 55 Kd, 65 Kd, and 110 Kd and mixtures thereof.

While this is one proposed method for isolation of the novel antigen ofthis invention other suitable methods may be used.

Once the antigen of this invention (ECA) is prepared, sera of suspectedceliac patients is added to well plates on which ECA has beenimmobilized. After incubation, excess antibody will be washed away and ahorseradish peroxidase labeled secondary antibody directed at humanIgA/IgG will be applied to the wells. The color changes will be read bythe ELISA spectrophotometer reader. The antigen ECA will bind to the IgAcontained in the sera of patients with Celiac Disease and this bindingconfirmed by the ELISA technique or procedures well known and describedin Hekkens W T, Van Twist M: “Physiological role of antigliadinantibodies and their appearance in Celiac Disease” and “SerologicDiagnosis of Celiac Disease”, Cleveland Press, 1990: 2A 21-58.

As above noted, in our search for a Celiac Disease antigen, we foundthat placenta tissue sections expressed an antigen which binds to theIgA contained in the sera of patients with Celiac Disease. We thenidentified, isolated and purified this antigen and designated thisprotein molecule as an Embroyonic Celiac Antigen (ECA).

Utilization of the newly discovered antigen: Develop a serologicdiagnostic test for Celiac Disease which can be easily performed andobjectively interpreted as a routine laboratory test. The isolated ECAprotein will be immobilized as an antigen on a 96 well plate. Sera frompatients suspected for Celiac Disease will be applied to the wells andassayed for the presence of reacting antibodies by Enzyme LinkedImmunoassay (ELISA). The ELISA technique will be developed as themethodology for utilizing the ECA antigen as a screening test for CeliacDisease.

We recently identified that an IgA protein in the sera of patients withCeliac Disease binds to an antigen expressed on placental tissuesections. In those cases of IgA deficient celiacs on IgG antibody wasfound to bind to the placenta sections. Microscopic evaluation ofplacental tissue sections indicate that the antigen was expressed onchorionic villi, specifically on their surface and within the cytoplasmof these cells. We then established a primary culture of placental cellline grown first in 24 well plates and subsequently expanded to a 102 cmtissue culture flask. The cells in culture were found to express theECA. An inhibition binding assay, however, demonstrated that the antigenwas not secreted into the culture media by the placental trophoblastcultured cell line. The antigen appears to be located in the cytoplasmand on the cell surface.

Collected placenta culture cells were lysed using lysis buffer. Proteinswere extracted from the culture lystate using ammonium sulfateprecipitation. After dialyzed with PBS, the precipitated proteins wereidentified by the SDS-PAGE gel electrophoresis. Proteins extracted fromthe placental cell culture lysate were applied to SDS-Page Gelelectrophoresis with a reducing and non-reducing condition reagent.Subsequently, proteins were transferred to nitrocellulose membrane andfinalized with Western Blotting procedures described in Laemmli, U.K.:Cleavage of structural proteins during the assembly of the head ofbacteriophage T4. Nature (London) 227:680, 1970.

Ten protein molecules were identified by the IgA contained in the seraof celiac patients. The molecular weights ranged from 17 Kd-110 Kd. Infurther experiments, using an inhibition assay, we proved that theisolated low molecular weight proteins (ranging 17 Kd-45 Kd) aregliadin-like polypeptides. These antigens were found to inhibit thebinding of the IgA/IgG in the sera of celiac patients to gliadinantigens. During further identification of the rest of the antigens, wefound two interesting proteins isolated from the placental cell linelysate. One exhibited a molecular weight of 110 Kd, and the other 65 Kd.These two proteins completely inhibited binding of IgA of Celiac Diseasepatients to rat kidney (Reticulin-like) and monkey esophagus(Endomysium-like). These two polypeptides were identified to haveisoelectricpoints of 5.1 and 5.8. Using high pressure liquidchromatography (HPLC) we were able to purify these two putativeEmbryonic Celiac Antigens. An inhibition assay indicated that the 110 Kdprotein inhibits at low concentrations binding of celiac patients' IgAto rat kidney. The 65 Kd protein, on the other hand, inhibitedcompletely the binding of IgA to the monkey esophagus. Both proteinmolecules had 60-70% of cross inhibition activity to either the rateliver or monkey esophagus. The 110 Kd protein is the heat labile proteinand tens to break down to a 55 Kd molecule without losing the antigenbinding activity.

We subsequently isolated the protein by the following two step isolationmethod. We first apply the crude extract protein isolated from thecultured placental cell lystate to a DEAE sephadex column and GelChromatography Sephacryl S-200 Hr Column (2.6×60 cm) and equilibrate itwith phosphate buffered saline pH 7.3. We purified the 110 Kd protein infraction collection number 40-45 and 65 Kd protein was purified infraction collection number 55-60. The fraction samples were collectedand purity was proofed by SDS-PAGE gel electrophoresis. Protein samplescollected were concentrated and frozen for further usage.

The primary placenta culture cell line was established in our laboratoryand was maintained at −70° C. until further usage. Using the freshplacental lysate additional 110 Kd was isolated. The frozen material, onthe other hand, yielded more of the 55 Kd molecule which also isidentified by the sera of celiac patients. The 110/55 Kd protein complexhas the capacity to inhibit 100% binding of the celiac sera of ratkidney. We therefore hypothesize that this is a reticulin-like antigen.This molecule complex also inhibits 60% of the binding activity ofceliac sera to the monkey esophagus. In distinction, the 65 Kd moleculeinhibits 100% binding of celiac sera to the monkey esophagus. However,this molecule inhibits only 60% binding activity of celiac sera to therat kidney. We therefore concluded that this is an endoymsial-likeantigen and designate it as the ECA.

The above putative Embryonic Celiac Antigens isolated from a culturedhuman placental cell line does not bind to the IgA or IgG contained inthe sera of normal patients, those with inflammatory bowel disease orother immunologic conditions.

1. Sample Preparation—starting with Human Placenta

a. Wash the cells twice with PBS.

b. Extract the washed cells with extraction buffer.

Extraction buffer: 10 mM Tris-HCl, PH7.2

0.15M NaCl, 0.02% NaNO₃ 5 mM Iodoacetamide

2% NP 40, ImM PMSF 5 mM MgCl₂

c. Centrifuge at 27000 g for 30 min, collect supernatant and store theextracts in liquid nitrogen.

2. SDS-Page

a. Add sodium dodecyl sulfate (SDS) sample buffer, with or without DTT,to the sample solution and boil the final sample solution in water bathfor 5 min.

b. Perform SDS-PAGE (polyacylamide gel electrophoresis) in a Laemmlisystem with slab gel containing 10% or 12% acrylamide. (Ref. Laemmli,Nature 227, 680-685, 1970)

c. Do silver staining with the gel. (Ref. Heukshoven, J. and Dermick. R,Electrophoresis, 6.103, 1985)

3. Western Immunoblot Analysis

a. Transfer the proteins to nitrocellulose membrane in Bio-RadTrans-Blot cell.

b. Stain the gel with Coomassie brilliant blue after the transfer toconfirm the uniformity of the transfer.

c. Block the membrane with the TBST buffer (containing 0.1% Tween 20 inPBS) for 1 hr. at room temperature.

d. Incubate the membrane with 1:500 dilution of CD patient serum (EMAtiter 1:6000) in TBST buffer for 2 hrs. at room temperature.

e. Wash the membrane three times with TBST.

f. Incubate the membrane with a 1:2000 dilution of the goat antihumanIgA conjugated to horseradish peroxidase (Sigma, A0295) in TBST for 2hrs. at room temperature.

g. Wash the membrane three times with TBST.

h. Incubate the membrane with ECL detection reagent for 1 min. andexpose to XAR5 X-ray film (Eastman Kodak, Rochester, N.Y.)

Ref:

(1) Towbin, H., T. Staehelin and J. Gordin, Proc. Natl. Acad. Sci., USA764350 (1979)

(2) Bryon Batteiger, et al J. of Immunol. Methods. 55, 297-307 (1982)

(3) Amersham Corporation ECL Western Blotting protocol booklet.

EXAMPLES Example 1

Placenta Cell Line Preparation

Placentas were discarded tissue obtained from the Pathology department.The placenta were perfused with collagenase buffer containing 0.05%trypsin after flushing with Hepes buffer. Single cell suspensions werecollected and different cell types were isolated and condensed from themixture into various subpopulations based in their densitycharacteristics using differential Percoll gradients. Cells from eachisolation zone were cultured in RPMI 1640 medium conditioned with 20%fetal bovine serum (FBS). Non-adherent cells were removed 12-24 hourslater.

Example 2

After rinsing with Hepes buffer, appropriate placental vasculature wascatheterized using a 16 gauge angiocath, perfused with Hepes buffer for½ hour, followed by a collaganase buffer digestion solution containing0.05% trypsin for one hour. The placental tissue rich in chorionic villiwas minced with scissors. The tissue mixture was allowed to digest whilebeing constantly stirred and incubated at the 37° C. for a minimum ofone hour. The singles cells were obtained by teasing apart the perfusedplacenta with forceps. The cells were pelleted by centrifugation (400×g)and washed twice with Fischer's medium.

Placental cells were obtained using density centrifugation withFicoll-Hypoque and discontinuous Percoll gradient. A discontinuousPercoll gradient was made by carefully layering 70%, 50%, 30% Percoll(vol/vol) with 1× Dulbecco's (Gibco) PBS in centrifuge tubes. Pelletedcells were resuspended in a 10 mL Fischer's medium layered over thePercoll gradients and centrifigured at 800×g for 30 minutes at roomtemperature.

Several zones appeared in the Percoll gradient: 0-30% trophoblast cells,Stroma cells, small vessels and villous fragments, connective tissueelements, 30-50% Polymorphonuclear leukocytes, 50-70% Red blood cells,and mononuclear cells: Pellet: Red Blood cells. The 0-30% interface wasseparated and washed twice with Fischer's medium. Cells were plated into25 cm² flasks with RPMI culture media containing 20% fetal bovine serum(FBS) and incubated at 37° C./5% Co₂/humidity-90% for 12-24 hours. Thenonadherent cells were removed from the culture. The adherent placentalcell line was propagated and expanded on 90 cm2 tissue culture flasksfor further experiments.

Example 3

The collected placental culture cells were lysed using lysis bufferwhich contained 10 mM tris base, 5 mM Mg Cl₂; 0.15M NaCl; 2% NP-40; 5 mMiodoacetamide, 0.02% NaNo₃, 1 mM PMSF (phenylmethylsulfonyl fluoride)and adjusted the Ph to 7.0.

To identified protein contained in the placental culture cell line, theprotein fraction was extracted from the placental cell culture lystateand applied to SDS-Page gel electrophoresis. The protein was applied toSDS-PAGE gel electrophoresis with a reducing and non-reducing conditionreagent. Subsequently protein was transferred to Nitrocellulose membraneand finalized with Western Blotting. Ten protein molecule antigens wereidentified by the IgA in the sera of patients with Celiac Disease; atmolecular weights ranging from of 17 Kd-110 Kd. In further experimentsusing an inhibition assay we proved that the isolated low molecularweight proteins (ranging 17 Kd-45 Kd) are gliadin-like polypeptides.These antigens were subsequently found to inhibit binding of the celiacpatient sera (IgA-IgG) to gliadin antigens. During furtheridentification of the rest of the antigens, we found 2 other proteinsisolated from the placental stromal cell line lystate. One exhibited amolecular weight of 110 Kd, and the other 65 Kd. These proteinscompletely inhibited binding of IgA of Celiac Disease patients to therat kidney (Reticulin like) and monkey esophagus (Endomysium like).These two polypeptides were identified to have isoelectricpoints of 5.1and 5.8. Using high pressure liquid chromatography we were able toseparate these two putative Embryonic Celiac Antigens. An inhibitionassay indicated that the 110 Kd protein inhibits completely at lowconcentrations binding of celiac patients IgA to rat kidney/liver. The65 Kd protein, on the other hand, inhibited completely the binding ofIgA to the monkey esophagus. Both protein molecules had 60-70% of crossinhibition activity to the other substrate, i.e., rat liver or monkeyesophagus. The 110 Kd protein is the heat labile protein and tends tobreak down to a 55 Kd molecule without losing the antigen bindingactivity.

Example 4

We subsequently isolated the protein by the following method from abovementioned placental culture lysate. Briefly, the method of isolationinvolves a two step isolation in which we use the crude extract proteinisolated from the cultured placental lystate. The protein contained inthe placenta lystate extract were first applied to a DEAE sephadexcolumn (1.0×10 cm) previously equilibrated with 0.05M tris/HCL, pH 7.4.The absorbed components were subsequently eluted with 0.05, 0.1, 0.2,0.3, 0.4, 0.5 M NaCl in 0.05M tris/HCL, pH7.4. The fractions werecollected (2.0 mL each) and concentrated using ultrafiltration (AmiconCentricon-3). The concentrated DEAE samples were applied to the SDS-PAGEgel electrophoresis for protein identification. 0.2 M in 0.05M tris/HDL,pH7.0 eluted a 110 Kd proteins, 0.5M NaCl in 0.05M tris/HCL, pH7.0eluted a 65 Kd major protein. Concentrated Chromatography proteinfraction were applied and identified by the SDS-PAGE gellelectrophoresis. DEAE chromatography collected samples were applied tothe Gel Chromatography using Sephacryl S-200 HR Column (2.6×70 cm) andequilibrated with phosphate buffered saline pH 7.3. Samples werecollected with 0.33 mL/min flow rate and 1 mL per tube. We purified the110 Kd protein in fraction collection number 40-45 and 65 Kd proteinwere purified in fraction collection number 55-60. The fraction sampleswere collected and the purity were proven by SDS-PAGE gelelectrophoresis. Purified and collected protein samples wereconcentrated and frozen for further usage.

The new antigen was isolated in our laboratory from a cell lineestablished from placenta tissue. Two examples of its efficacy are: (1)indirect immunofluorescence staining indicates that this cell lineexpresses the antigen which binds to antibodies found in the sera ofceliac patients, (2) the protein isolated from the crude extract of theprimary placenta cell line binds as demonstrated by electrophoresis andWestern Blotting to the IgA antibodies in the sera of celiac patients.0.25 mg of the isolated ECA having a molecular weight of 65 Kd willinhibit binding of celiac patients serum to monkey esophagus or liver.On the other hand, 0.25 mg of isolated ECA having a molecular weight of110/55 Kd will inhibit binding of celiac patients sera to rat kidney orliver but will not inhibit binding to monkey esophagus.

The screening process: Since the clinical presentation of Celiac Diseaseis quite variable, patients with symptoms such as chronic diarrhea,bloating, abdominal distention, short stature, growth disturbances anddiabetes may require screening. Early diagnosis is important sincetreatment with a special gluten-free diet seems to reduce the risk ofsubsequent gastrointestinal malignancy. The diagnosis of Celiac Diseasehas required intestinal biopsy. A reliable noninvasive test for thedisease has long been sought. The isolated ECA which binds the sera ofCeliac Disease patients is a promising screening test. The ECA will beimmobilized on 96 well plates. The sera of suspected celiac patientswith the above symptoms will be added. After incubation, excess antibodywill be washed away and a horseradish peroxidase labeled secondaryantibody directed at human IgA/IgG will be applied to the wells. Thecolor changes will be read by the spectrophotometer.

Below is a general outline of the procedure for producing the novelantigen of this invention:

1. Primary Placenta Cell Culture:

Wash, perfuse and digest placenta tissue with hepes buffer andcollagenase buffer—single cell suspension preparation.

2. Enrichment and concentration of the adherent cells from the cellsuspension using discontinuous percoll gradient.

3. Protein extraction from the primary placental cell line using anextraction buffer and ultra-centrifugation.

4. Embryonic Celiac Antigen (ECA) Identification: Placental cellextracts were applied to SDS-PAGE gel electrophoresis to separate theproteins. Separated proteins were transferred to the nitrocellulosemembrane (Western Blotting) incubated with the serum of patients withCeliac Disease. Horseradish-peroxidase conjugated rabbit anti-human IgAis applied to identify the binding of celiac patients IgA to theantigen. Ten monocomponent polypeptides were detected with CeliacDisease sera on nitrocellulose membrane. Three single polypeptides withmolecular weight higher than 55 Kd (55 Kd-65 Kd and 110 Kd) andIsoelectric points 5.1-5.8 demonstrated inhibition of binding of celiacsera on the monkey esophagus and rat kidney/liver. Seven othermonopolypeptides with molecular weights of less than 55 Kd did notinhibit binding.

5. Embryonic Celiac Antigen (ECA) Isolation: Initial separation ofplacental primary culture cell extract were done by injecting to thegell filtration (size exclusion) of HPLC Column from Bekman (UltraSphero Gell” (SEC 3000) size 5 micron. The column size is 7.5 mM×300 mM.Competition binding assay was used to identify the proteins. Separatedproteins were also applied to the SDS-PAGE and Western Blotting toidentify the purity and molecular weight of the proteins.

6. Embryonic Celiac Antigen (ECA) Isolation (Large volume separationusing the gel chromatography): Separation is performed using DEAE ionexchange chromatography and Sephacryl S-200 HR Column (2.6×) cm andequilibrate with phosphate buffered saline pH7.3.

Enzyme Link Immuno Assay (ELISA) Test Kit Development

ECA, the protein molecules reacting with Celiac Disease IgA and IgG wereseparated and purified from the primary culture of placental stromalcells. These proteins were coated to the 96 well plate. Serum from testpatients will be applied to the plate and a secondaryHorseradish-Peroxidase anti-human IgA antibody will be applied. Plateswill be read using ELISA spectrophotometry reader.

A step-by-step procedure for the antigen ECA preparation is as follows:

(1) Primary Placenta Cell Culture:

Wash, perfuse, and digest placenta tissue with hepes buffer andcollagenase buffer—Single cell suspension preparation.

(2) Enrichment and Concentration of the adherent cells from the cellsuspension using discontinuous percoll gradient.

(3) Protein extraction from the primary placental cell line using anextraction buffer and ultra-centrifugation.

(4) Placental cell extracts were applied to the SDS-PAGE GelElectrophoresis to separate the proteins.

(5) Separated protein were transferred to the nitrocellulose membrane(Western Blotting) incubated with the serum of patients with CeliacDisease.

(6) Horseradish-peroxidase conjugated mouse anti human IgA is applied toidentify the binding of celiac patients IgA to the antigen. (10monocomponent polypeptides could be identified by the IgA of sera fromCeliac Disease patients on nitrocellulose membrane.)

(7) Separation of primary culture cells extract was done by injectingprotein extract to the Gel filtration (size exclusion) of HPLC Columnfrom Bekman “Ultra Sphero Gell” (SEC 3000) size 5 micron. The columnsize is 7.5 mM×300 mM.

(8) Competition biding assay was used to identify the separated proteins(Three single polypeptides with molecular weight higher than 55 Kd (55Kd-65 Kd and 110 Kd) and Isoelectric point 5.1-5.8 demonstratedinhibition of binding of Celiac Disease patient serum IgA/IgG on themonkey esophagus and rat kidney/liver. Seven other monopolypeptides withmolecular weight of less than 55 Kd inhibit binding to Gliadin).

(9) Large volume separation Using the Gel Chromatography Separation isperformed using DEAE sephadex column followed by the Sephacryl GelChromatography S-200 HR Column, bed size dimension are 2.6×32 Cm.

Enzyme Link Immuno Assay (ELISA) Test Kit Development ECA, the proteinmolecules reacting with Celiac Disease IgA and IgG were separated andpurified from the primary culture of Placental stromal cells. ECA willbe coated to the 96 well plate. Serum from test patients will be appliedto the plate and a secondary Horseradish-Peroxidase anti-human IgAantibody will be applied. Plate will be read using ELISAspectrophotometry reader.

The preferred and optimumly preferred embodiments of the presentinvention have been described herein and shown in the accompanyingspecification to illustrate the underlying principles of the invention,but it is to be understood that numerous modifications and ramificationsmay be made without departing from the spirit and scope of thisinvention.

What is claimed is:
 1. An isolated antibody-antigen complex prepared bya process comprising contacting (1) an embryonic celiac antigen isolatedby extraction from a cultured human placenta cell, said antigencomprising protein molecules with an isoelectric point (pI) range offrom 5.1-5.8 and an apparent molecular weight selected from the groupconsisting of 55 Kd, 65 Kd, and 110 Kd, with (2) sera from a celiacdisease patient.
 2. The isolated antibody-antigen complex according toclaim 1 wherein said antigen comprises protein molecules with anapparent molecular wight of 65 Kd.